Development of DQ-MHC Class II Monoclonal Antibodies—A New, Targeted Therapeutic Approach in Type 1 Diabetes

Purpose: Type 1 diabetes (T1D) is a disease affecting almost three million Americans; every year 30,000 new cases are diagnosed, 50% of whom are children. There is currently no curative therapy for T1D and the only available treatment is insulin replacement. Substantial recent data demonstrate a strong association between the MHC Class II molecule DQ8 and the development of T1D. Indeed, DQ8 has been shown to present antigenic peptides (such as InsB:9-23 and GAD65) driving activation of CD4+ T cells in T1D patients. The goal of this study was to identify and optimize a monoclonal antibody that binds to DQ8 molecule and blocks the continued activation of DQ8 restricted T cells.

Methods: Hybridoma clones were generated from spleens of BALB/c mice immunized with DQ8 molecule bound with the InsB:9-23 peptide and screened by high throughput flow cytometry using human B-cells expressing DQ8. 28 clones found positive by flow cytometry were functionally tested in a mixed lymphocyte reaction containing a murine T cell line expressing a human TCR specific for the InsB: 9-23 - DQ8 complex. DQ8-103, a murine version of our best candidate antibody (DQ8-27), was tested in transgenic DQ8 mice immunized with GAD65 peptide; Cytokine production from the recall response ex vivo and in vivo was evaluated using a Luminex assay. Immunophenotyping of blood, spleens and draining lymph nodes isolated from immunized DQ8 mice was done by mass cytometry (CyTOF).

Results: We generated a monoclonal antibody against DQ8 (DQ8-27), that was able to bind human DQ8 molecule expressed on human B-cells and on murine splenocytes isolated from transgenic DQ8 mice. The murine version of DQ8-27 (DQ8-103) was able to significantly inhibit T cell activation in DQ8 mice immunized with GAD65 both ex vivo and in vivo, by decreasing the production of pro inflammatory cytokines IL-2 and IFN-γ. Of note, DQ8-103 caused a significant depletion of DQ8+ B-cells in treated mice compared to mice injected with a control antibody.